Plant Cell Reports

, Volume 28, Issue 3, pp 429–444 | Cite as

Efficient, reproducible Agrobacterium-mediated transformation of sorghum using heat treatment of immature embryos

  • Songul Gurel
  • Ekrem Gurel
  • Rajvinder Kaur
  • Joshua Wong
  • Ling Meng
  • Han-Qi Tan
  • Peggy G. Lemaux
Genetic Transformation and Hybridization


A number of parameters related to Agrobacterium-mediated infection were tested to optimize transformation frequencies of sorghum (Sorghum bicolor L.). A plasmid with a selectable marker, phosphomannose isomerase, and an sgfp reporter gene was used. First, storing immature spikes at 4°C before use decreased frequency of GFP-expressing calli, for example, in sorghum variety P898012 from 22.5% at 0 day to 6.4% at 5 days. Next, heating immature embryos (IEs) at various temperatures for 3 min prior to Agrobacterium infection increased frequencies of GFP-expressing calli, of mannose-selected calli and of transformed calli. The optimal 43°C heat treatment increased transformation frequencies from 2.6% with no heat to 7.6%. Using different heating times at 43°C prior to infection showed 3 min was optimal. Centrifuging IEs with no heat or heating at various temperatures decreased frequencies of all tissue responses; however, both heat and centrifugation increased de-differentiation of tissue. If IEs were cooled at 25°C versus on ice after heating and prior to infection, numbers with GFP-expressing cells increased from 34.2 to 49.1%. The most optimal treatment, 43°C for 3 min, cooling at 25°C and no centrifugation, yielded 49.1% GFP-expressing calli and 8.3% stable transformation frequency. Transformation frequencies greater than 7% were routinely observed using similar treatments over 5 months of testing. This reproducible frequency, calculated as numbers of independent IEs producing regenerable transgenic tissues, confirmed by PCR, western and DNA hybridization analysis, divided by total numbers of IEs infected, is several-fold higher than published frequencies.


Agrobacterium Heat treatment Phosphomannose isomerase Sorghum Transformation 



S. Gurel (S.G.) thanks The General Directorate of Turkish Sugar Factories Co. for the work permission abroad. E. Gurel (E.G.) deeply appreciates the financial support of TUBITAK (The Scientific and Technological Research Council of Turkey). E.G. and S.G. are also cordially grateful to Professor P.G. Lemaux (P.G.L.) for the opportunity to come to the University of California, Berkeley, as visiting scholars. P.G.L. was supported by USDA Cooperative Extension through the University of California. R. Kaur and J. Wong were supported through a Gates Foundation Grand Challenges for Global Health award to Africa Harvest, Nairobi, Kenya. H.-Q. Tan was supported by the UC Berkeley College of Natural Resources Sponsored Projects for Undergraduate Research. The authors thank Dr. George Liang for the gift of the pGFP-PMI plasmid, Toshihiko Komari and Naoki Takemori from Japan Tobacco Inc., for helpful discussions, Dr. Lixin Zhu at UC Berkeley for the chemiluminescence analysis of GFP and Eric Trieu for technical help with PCR analyses.


  1. An G, Ebert PR, Mitra A, Ha SB (1988) Binary vectors. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual, Kluwer Academic Publishers, Dordrecht, pp A31-A3/19Google Scholar
  2. Arriola PE, Ellstrand NC (1997) Fitness of interspecific hybrids in the genus Sorghum: persistence of crop genes in wild populations. Ecol Appl 7:512–518CrossRefGoogle Scholar
  3. Aswath CR, Mo SY, Kim DH, Park SW (2006) Agrobacterium and biolistic transformation of onion using non-antibiotic selection marker phosphomannose isomerase. Plant Cell Rep 25:92–99PubMedCrossRefGoogle Scholar
  4. Cai T, Butler L (1990) Plant regeneration from embryogenic callus initiated from immature inflorescences of several high-tannin sorghums. Plant Cell Tissue Organ Cult 20:101–110CrossRefGoogle Scholar
  5. Cai T, Daly B, Butler L (1987) Callus induction and plant regeneration from shoot portions of mature embryos of high-tannin sorghum. Plant Cell Tissue Organ Cult 9:245–252CrossRefGoogle Scholar
  6. Carvalho CHS, Zehr UB, Gunaratna N, Anderson J, Kononowicz HH, Hodges TK, Axtell JD (2004) Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency. Genet Mol Biol 27:259–269Google Scholar
  7. Casas AM, Kononowicz AK, Zehr UB, Tomes DT, Axtell JD, Butler LG, Bressan RA, Hasegawa PM (1993) Transgenic sorghum plants via microprojectile bombardment. Proc Natl Acad Sci USA 90:11212–11216PubMedCrossRefGoogle Scholar
  8. Casas AM, Kononowicz AK, Haan TG, Zhang L, Tomes DT, Bressan RA, Hasegawa PM (1997) Transgenic sorghum plants obtained after microprojectile bombardment of immature inflorescences. In Vitro Cell Dev Biol Plant 33:92–100Google Scholar
  9. Center for Food Safety and Applied Nutrition, Food and Drug Administration (1998) Guidance for industry: use of antibiotic resistance marker genes in transgenic plants.
  10. Chiu W, Hiwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J, Chiu WL (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330PubMedCrossRefGoogle Scholar
  11. Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Trans Res 5:213–218CrossRefGoogle Scholar
  12. Dicko MH, Gruppen H, Traore AS, Voragen AGJ, Berkel WJH (2006) Phenolic compounds and related enzymes as determinants of sorghum for food use. Biotech Mol Biol Rev 1:21–38Google Scholar
  13. Dykes L, Rooney LW (2006) Sorghum and millet phenols and antioxidants. J Cereal Sci 44:236–251CrossRefGoogle Scholar
  14. Ellstrand NC, Prentice HC, Hancock JF (1999) Gene flow and introgression from domesticated plants into their wild relatives. Annu Rev Ecol Syst 30:539–556CrossRefGoogle Scholar
  15. Emani C, Sunilkumar G, Rathore KS (2002) Transgene silencing and reactivation in sorghum. Plant Sci 162:181–192CrossRefGoogle Scholar
  16. Gao Z, Jayaraj J, Muthukrishnan S, Claflin L, Liang GH (2005a) Efficient genetic transformation of sorghum using a visual screening marker. Genome 48:321–333PubMedGoogle Scholar
  17. Gao Z, Xie X, Ling Y, Muthukrishnan S, Liang GH (2005b) Agrobacterium tumefaciens-mediated sorghum transformation using a mannose selection system. Plant Biotech J 3:591–599CrossRefGoogle Scholar
  18. Girijashankar V, Sharma HC, Sharma KK, Swathisree V, Prasad LS, Bhat BV, Royer M, Secundo BS, Narasu ML, Altosaar I, Seetharama N (2005) Development of transgenic sorghum for insect resistance against the spotted stem borer (Chilo partellus). Plant Cell Rep 24:513–522PubMedCrossRefGoogle Scholar
  19. Hagio T, Blowers AD, Earle ED (1991) Stable transformation of sorghum cell cultures after bombardment with DNA-coated microprojectiles. Plant Cell Rep 10:260–264CrossRefGoogle Scholar
  20. Hansen G (1998) Plant transformation methods. US Patent Number 6, 162, 965Google Scholar
  21. Hansen G (2000) Evidence for Agrobacterium-induced apoptosis in maize cells. Mol Plant Microbe Interact 13:649–657PubMedCrossRefGoogle Scholar
  22. He Z, Duan Z, Liang W, Chen F, Yao W, Liang H, Yue C, Sun Z, Chen F, Dai J (2006) Mannose selection system used for cucumber transformation. Plant Cell Rep 25:953–958PubMedCrossRefGoogle Scholar
  23. Hiei Y, Ishida Y, Kasaoka K, Komari T (2006) Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection with Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult 87:233–243CrossRefGoogle Scholar
  24. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoot RA (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180CrossRefGoogle Scholar
  25. Hood EE, Helmer GD, Fraley RT, Chilton MD (1986) The hypovirulence of Agrobacterium tumefaciens A281 is encoded in the region of pTiBo542 outside the T-DNA. J Bacteriol 168:1291–1301PubMedGoogle Scholar
  26. Howe A, Sato S, Dweikat I, Fromm M, Clemente T (2006) Rapid and reproducible Agrobacterium-mediated transformation of sorghum. Plant Cell Rep 25:784–791PubMedCrossRefGoogle Scholar
  27. Iordanskjy S, Zhao Y, Dubrovsky L, Iordanskaya T, Chen M, Liang D, Bukrinsky M (2004) Heat shock protein 70 protects cells from cell cycle arrest and apoptosis induced by human immunodeficiency virus type 1 viral protein R. J Virol 78:9697–9704CrossRefGoogle Scholar
  28. Jain M, Chengalrayan K, Abouzid A, Gallo M (2007) Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugar cane (Saccharum spp. hybrid) plants. Plant Cell Rep 26:581–590PubMedCrossRefGoogle Scholar
  29. Jenks MA, Joly RJ, Peters PJ, Rich PJ, Axtell JD, Ashworth EN (1994) Chemically induced cuticle mutation affecting epidermal conductance to water vapor and disease susceptibility in Sorghum bicolor (L.) Moench. Plant Physiol 105:1239–1245PubMedGoogle Scholar
  30. Joersbo M, Donaldson I, Kreiberg J, Petersen SG, Brunstedt J, Okkels FT (1998) Analysis of mannose selection used for transformation of sugar beet. Mol Breeding 4:111–117CrossRefGoogle Scholar
  31. Kaeppler HF, Pederson JF (1997) Evaluation of 41 elite and exotic inbred sorghum genotypes for high quality callus production. Plant Cell Tissue Organ Cult 48:71–75CrossRefGoogle Scholar
  32. Khanna H, Becker D, Kleidon J, Dale J (2004) Centrifugation-assisted Agrobacterium tumefaciens-mediated transformation (CAAT) of embryogenic cell suspensions of banana (Musa spp. Cavendish AAA and Lady finger Aab). Mol Breeding 14:239–252CrossRefGoogle Scholar
  33. Lemaux PG (2008) Genetically engineered plants and foods. A scientist’s analysis of the issues. Part I. Ann Rev Plant Biol 59:771–812CrossRefGoogle Scholar
  34. Li HQ, Kang PJ, Li ML, Li MR (2007) Genetic transformation of Torenia fournieri using the PMI/mannose selection system. Plant Cell Tissue Organ Cult 90:103–109CrossRefGoogle Scholar
  35. Masteller VJ, Holden DJ (1970) The growth of and organ formation from callus tissue of sorghum. Plant Physiol 45:362–364PubMedCrossRefGoogle Scholar
  36. McElroy D, Louwerse JD, McElroy SM, Lemaux PG (1997) Development of a simple transient assay for Ac/Ds activity in cells of intact barley tissue. Plant J 11:157–165PubMedCrossRefGoogle Scholar
  37. Miles H, Guest JR (1984) Nucleotide sequence and transcriptional start point of the phosphomannose isomerase gene (manA) of Escherichia coli. Gene 32:41–48PubMedCrossRefGoogle Scholar
  38. Miller FR (1984) Registration of RTx430 sorghum parental line. Crop Sci 24:1224CrossRefGoogle Scholar
  39. Min BW, Cho YN, Song MJ, Noh TK, Kim BK, Chae WK, Park YS, Choi YD, Harn CH (2007) Successful genetic transformation of Chinese cabbage using phosphomannose isomerase as a selection marker. Plant Cell Rep 26:337–344PubMedCrossRefGoogle Scholar
  40. Morrell PL, Williams-Coplin TD, Lattu AL, Bowers JE, Chandler JM, Paterson AH (2005) Crop-to-weed introgression has impacted allelic composition of johnsongrass populations with and without recent exposure to cultivated sorghum. Mol Ecol 4:2143–2154CrossRefGoogle Scholar
  41. MSTATC (2004) MSTATC, Version 1.42, Michigan State University, USAGoogle Scholar
  42. Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20:87–90PubMedCrossRefGoogle Scholar
  43. Nguyen TV, Thu TT, Claeys M, Angenon G (2007) Agrobacterium-mediated transformation of sorghum [Sorghum bicolor (L.) Moench] using an improved in vitro regeneration system. Plant Cell Tissue Organ Cult 91:155–164CrossRefGoogle Scholar
  44. O’Kennedy MM, Grootboom A, Shewry PR (2006) Harnessing sorghum and millet biotechnology for food and health. J Cereal Sci 44:224–235CrossRefGoogle Scholar
  45. Penna S, Sági L, Swennen R (2002) Positive selectable marker genes for routine plant transformation. In Vitro Cell Dev Biol Plant 38:125–128CrossRefGoogle Scholar
  46. Privalle LS (2002) Phosphomannose isomerase, a novel plant selection system. Ann N Y Acad Sci 964:129–138PubMedGoogle Scholar
  47. Ramesh SA, Kaiser BN, Franks T, Collins G, Sedgley M (2006) Improved methods in Agrobacterium-mediated transformation of almond using positive (mannose/pmi) or negative (kanamycin resistance) selection-based protocols. Plant Cell Rep 25:821–828PubMedCrossRefGoogle Scholar
  48. Seetharama N, Sairam RV, Rani TS (2000) Regeneration of sorghum from shoot tip cultures and field performance of the progeny. Plant Cell Tissue Organ Cult 61:169–173CrossRefGoogle Scholar
  49. Sheen J, Hwang S, Niwa Y, Kobayashi H, Galbraith DW (1995) Green-fluorescent protein as a new vital marker in plant cells. Plant J 8:777–784PubMedCrossRefGoogle Scholar
  50. Shrawat HK (2007) Genetic transformation of cereals mediated by Agrobacterium. Inform Syst Biotechnol News Rep, February, pp 8–10.
  51. Shrawat HK, Lörz H (2006) Agrobacterium-mediated transformation of cereals: a promising approach crossing barriers. Plant Biotechnol J 4:575–603PubMedCrossRefGoogle Scholar
  52. Singh J, Zhang S, Chen C, Cooper L, Bregitzer P, Sturbaum A, Hayes PM, Lemaux PG (2006) High-frequency Ds remobilization over multiple generations in barley facilitates gene tagging in large genome cereals. Plant Mol Biol 62:937–950PubMedCrossRefGoogle Scholar
  53. Tadesse Y, Sagi L, Swennen R, Jacobs M (2003) Optimization of transformation conditions and production of transgenic sorghum (Sorghum bicolor) via microprojectile bombardment. Plant Cell Tissue Organ Cult 75:1–18CrossRefGoogle Scholar
  54. Thompson CJ, Movva NR, Tizard R, Crameri R, Davies JE, Lauwereys M, Botterman J (1987) Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO J 6:2519–2523PubMedGoogle Scholar
  55. Wenck A, Hansen G (2004) Positive selection. In: Pena L (ed) Transgenic plants: methods and protocols. Humana Press, Totowa, pp 227–235CrossRefGoogle Scholar
  56. Wright M, Dawson J, Dunder E, Suttie J, Reed J, Kramer C, Chang Y, Novitzky R, Wang H, Artim-Moore L (2001) Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker. Plant Cell Rep 20:429–439CrossRefGoogle Scholar
  57. Zhao ZY (2006) Sorghum (Sorghum bicolor L.). In: Wang K (ed) Methods in molecular biology. Agrobacterium protocols 2/e, vol 1. Humana Press, Totowa, 343:233–244Google Scholar
  58. Zhao ZY, Cai T, Tagliani L, Miller M, Wang N, Pang H, Rudert M, Schroeder S, Hondred D, Seltzer J, Pierce D (2000) Agrobacterium-mediated sorghum transformation. Plant Mol Biol 44:789–798PubMedCrossRefGoogle Scholar
  59. Zhao ZY, Glassman K, Sewalt V, Wang N, Miller M, Chang S, Thompson T, Catron S, Wu E, Bidney D, Kedebe Y, Jung R (2003) Nutritionally improved transgenic sorghum. In: Vasil IK (ed) Plant Biotechnology 2002 and beyond. Kluwer Academic Publishers, Dordrecht, pp 413–416Google Scholar
  60. Zhu H, Muthukrishnan S, Krishnaveni S, Wilde G, Jeoung JM, Liang GH (1998) Biolistic transformation of sorghum using a rice chitinase gene. J Genet Breeding 52:243–252Google Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Songul Gurel
    • 1
    • 2
  • Ekrem Gurel
    • 1
    • 3
  • Rajvinder Kaur
    • 1
  • Joshua Wong
    • 1
  • Ling Meng
    • 1
  • Han-Qi Tan
    • 1
  • Peggy G. Lemaux
    • 1
  1. 1.Department of Plant and Microbial BiologyUniversity of CaliforniaBerkeleyUSA
  2. 2.Department of Plant BreedingSugar InstituteAnkaraTurkey
  3. 3.Department of Biology, Faculty of Science and LiteratureAbant Izzet Baysal UniversityBoluTurkey

Personalised recommendations